The Journal of Antibiotics (2012) 65, 593–598

& 2012 Japan Antibiotics Research Association All rights reserved 0021-8820/12
www.nature.com/ja

ORIGINAL ARTICLE

Activity of the thiopeptide antibiotic nosiheptide

against contemporary strains of methicillin-resistant
Staphylococcus aureus
Nina M Haste1, Wdee Thienphrapa1, Dan N Tran1, Sandra Loesgen2,5, Peng Sun2, Sang-Jip Nam2,4,
Paul R Jensen2, William Fenical2,3, George Sakoulas1, Victor Nizet1,3 and Mary E Hensler1

The rapid rise in antimicrobial resistance in bacteria has generated an increased demand for the development of novel therapies
to treat contemporary infections, especially those caused by methicillin-resistant Staphylococcus aureus (MRSA). However,
antimicrobial development has been largely abandoned by the pharmaceutical industry. We recently isolated the previously
described thiopeptide antibiotic nosiheptide from a marine actinomycete strain and evaluated its activity against contemporary
clinically relevant bacterial pathogens. Nosiheptide exhibited extremely potent activity against all contemporary MRSA strains
tested including multiple drug-resistant clinical isolates, with MIC values p0.25 mg l 1. Nosiheptide was also highly active
against Enterococcus spp. and the contemporary hypervirulent BI/NAP1/027 strain of Clostridium difficile but was inactive
against most Gram-negative strains tested. Time-kill analysis revealed nosiheptide to be rapidly bactericidal against MRSA in
a concentration- and time-dependent manner, with a nearly 2-log kill noted at 6 h at 10  MIC. Furthermore, nosiheptide was
found to be non-cytotoxic against mammalian cells at 44100  MIC, and its anti-MRSA activity was not inhibited by 20%
human serum. Notably, nosiheptide exhibited a significantly prolonged post-antibiotic effect against both healthcare- and
community-associated MRSA compared with vancomycin. Nosiheptide also demonstrated in vivo activity in a murine model of
MRSA infection, and therefore represents a promising antibiotic for the treatment of serious infections caused by contemporary
strains of MRSA.
The Journal of Antibiotics (2012) 65, 593–598; doi:10.1038/ja.2012.77; published online 10 October 2012

Keywords: contemporary MRSA; marine actinomycete; nosiheptide; thiopeptide

INTRODUCTION multhiomycin, was originally isolated in 1970 and shown to be

Multidrug resistant hospital-associated (HA-) and highly virulent
community-associated (CA-) methicillin-resistant Staphylococcus
aureus (MRSA) have shown increased tolerance or resistance in
recent years to vancomycin, linezolid and daptomycin with accom-
panying reduction in clinical antimicrobial efficacy. The development
of novel agents without cross-resistance to current antimicrobials
against MRSA infections is desperately needed.1
In our screen of marine-derived actinomycete extract libraries for
anti-MRSA activity, we identified a potent fraction derived from
strain CNT-373, a Streptomycetes species isolated from a marine
sediment collected in Fiji. The 1221.16-mol wt active component was
purified and identified by NMR as the thiopeptide antibiotic
nosiheptide. Thiopeptide antibiotics encompass a large family of
compounds that include thiostrepton and nocathiacin and are
comprised of sulfur- and nitrogen-rich heterocycles linked to non-
natural amino acids.2,3 Nosiheptide, also referred to historically as
structurally similar to thiostrepton.4,5 Several thiopeptide antibiotics
including nosiheptide block protein synthesis by the inhibition of
elongation factors Tu and G.6 Historically, nosiheptide has been used
as a growth-promoting additive in animal feed7 but was never
developed further as a human therapeutic.
Although the discovery of new chemical scaffolds provides the
potential for novel drugs with new mechanisms of action, the
re-investigation of previously discovered antibacterial scaffolds also
provides a valuable source of compounds with therapeutic potential.
Surprisingly, despite the structural identification and description of
Gram-positive antibacterial activity, no detailed characterization of
nosiheptide activity against contemporary drug-resistant strains such
as MRSA has been undertaken. Here, we investigate nosiheptide
activity in vitro against a panel of contemporary MRSA and other
Gram-positive clinical isolates. MRSA killing kinetics and post-
antibiotic effects (PAE) are characterized, and nosiheptide in vivo

1Department of Pediatrics, University of California San Diego, La Jolla, CA, USA; 2Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography,
University of California San Diego, La Jolla, CA, USA and 3Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA
Correspondence: Dr ME Hensler, Department of Pediatrics, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA.
E-mail: mhensler@ucsd.edu
4Current address: College of Pharmacy, Suncheon National University, Suncheon 540-950, Republic of Korea.
5Current address: Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA 20892.

Received 6 June 2012; revised 9 August 2012; accepted 15 August 2012; published online 10 October 2012
 Nosiheptide activity against contemporary MRSA
NM Haste et al
594

activity is demonstrated. Our results indicate that ‘re-discovered’ Table 1 1H and 13C NMR spectral data of nosiheptide (d in p.p.m.,
natural product antibiotics may harbor meaningful therapeutic J in Hz)
activity against contemporary multidrug resistant pathogens, and Ind CO 180.69 But 3 128.14 6.32 (q, J ¼ 6.84)
therefore warrant further consideration and preclinical development Glu CO 173.52 Pyr 4 127.17 7.64 (s)
in hopes of expanding our current limited pharmacological arsenal. Thz(3) 2 170.18 Thz(5) 5 126.52 8.28 (s)
Thz(4) 2 169.64 Thz(1) 5 125.82 8.47 (s)
MATERIALS AND METHODS Thr CO 169.44 Thz(3) 5 125.67 8.18 (s)
Strain isolation and identification Thz(5) 2 166.83 Ind 6 125.28 7.41 (t, J ¼ 7.57)
Strain CNT-373 was isolated from a marine sediment sample collected at a Thz(2) 2 166.41 Ind 3a 124.36
depth of 5 m off Nacula Island, Fiji. The sediment was dried overnight in a Deala CO 165.99 Thz(2) 5 123.91 7.91 (s)
laminar flow hood and then stamped on agar plates containing medium A1 Thz(1) 2 164.85 Ind 5 122.24 7.12 (d, J ¼ 6.95)
(10 g starch, 4 g yeast extract, 2 g peptone, 16 g agar and 1 l seawater) Thz(3) CO 161.43 Thz(4) 5 120.38 7.66 (s)
supplemented with 100 mg l 1 cyclohexamide to reduce fungal growth. The Thz(2) CO 160.84 Ind 3 118.90
strain was identified as a Streptomyces sp. based on 16S rRNA gene sequence Thz(1) CO 160.57 Ind 7 115.31 7.79 (d, J ¼ 7.04)
analysis (http://eztaxon-e.ezbiocloud.net/ezt_identify) and shares greatest simi- Thz(5) CO 158.59 Deala 3 104.03 6.60 E (d, J ¼ 1.89),
larity (98.3%) with the type strain Streptomyces althioticus. This low level of 5.58 Z (d, J ¼ 1.89)
sequence identity suggests it may be a new species. The 16S rRNA sequence has
Thz(4) 4 155.00 Thr 3 67.45 4.04 (m)
been deposited in GenBank under accession number JQ946086.
Pyr 3 150.30 Glu 4 67.18 4.14 (d, J ¼ 11.54)
Thz(1) 4 149.81 Ind 4’ 66.60 5.85 (d, J ¼ 11.25),
Fermentation and extraction 5.02 (d, br, J ¼ 10.79)
A 2-ml-frozen glycerol stock of strain CNT-373 was used to inoculate 25 ml of Thz(5) 4 149.76 Thr 2 56.75 4.36 (d, J ¼ 7.79)
liquid A1 medium and shaken at 230 r.p.m. and 27 1C. After 5 days, the seed Thz(3) 4 148.45 Cys 2 50.17 5.98 (d, br, J ¼ 9.00)
culture was used to inoculate a 1-l culture in growth medium A1bfe þ C (A1
Thz(2) 4 147.86 Glu 2 45.85 5.76 (t, J ¼ 10.19)
medium with the addition of 1 g CaCO3, 100 mg KBr and 40 mg Fe2(S-
Pyr 6 143.99 Glu 3 36.72 2.35 S (m), 1.80 R (m)
O4)34H2O). After 3 days of shaking, 25-ml aliquots were used to inoculate 18
Ind 7a 137.44 Cys 3 29.71 3.79 (dd, J ¼ 3.15, 12.06),
Fernbach flasks (2.8 l) each containing 1 l medium A1bfe þ C. After 7 days of
3.71 (dd, J ¼ 4.50, 12.00)
cultivation, sterilized XAD-16 resin (20 g l 1) was added, shaken for 6 h and
Pyr 2 134.46 Thr CH3 17.52 0.95 (s)
collected by filtration through cheesecloth. The resin was then washed with
deionized water and eluted with acetone. The acetone solvent was removed Deala 2 132.94 But CH3 14.58 1.68 (d, J ¼ 7.00)
under reduced pressure and the resulting aqueous layer extracted with ethyl Ind 2 130.64 Ind CH3 11.96 2.54 (s)
acetate (3  500 ml). The combined ethyl acetate extracts were dried over Pyr 5 130.44 Ind NH 10.83 (s, br)
anhydrous sodium sulfate, decanted and concentrated to dryness to yield 2.4 g But 2 129.41 Deala NH 9.99 (s)
of crude material. Ind 4 128.73 But NH 9.76 (s)
Glu NH 7.97 (d, br, J ¼ 7.29)
Isolation of nosiheptide Glu OH not observed
The crude extract was dissolved in methanol and dichloromethane, adsorbed Cys NH 7.82 (s)
onto silica gel (2.5 g), and fractionated on a short column of silica gel Thr NH 7.50 (s)
(6  2.5 cm, h  w) using a 10% step gradient from 100% isooctane to 100% Thr OH 7.48 (s)
ethyl acetate as eluents. Fractions with antibacterial activity were eluted with 90
and 100% ethyl acetate. The active fractions were pooled and subjected to a
size exclusion column chromatography with Sephadex LH-20 using methanol
as eluent. The final purification step was achieved with silicia gel, using ethyl
acetate with 10% dimethylformamide to give nosiheptide (210 mg).

Spectroscopic analysis of nosiheptide

1H, 13C and 2D NMR spectroscopic data were obtained on a Varian Inova 500-

MHz spectrometer in a solvent mixture of methanol-d4 and CDCl3 (3:1) to

facilitate solubility. Offline processing was conducted using topspin NMR
software by Bruker BioSpin 2011 (iNMR, http://www.inmr.net). The NMR
data (Table 1) are in good agreement with previously published NMR data for
nosiheptide,8 with the resultant nosiheptide structure shown (Figure 1). High-
resolution ESI-TOF mass spectra were provided by the MS facility at the
Department of Chemistry and Biochemistry at the University of California
San Diego, CA: HR-ESI-TOF-MS [M þ H] þ m/z 1222.1565 (calcd for
C51H44N13O12S6 1222.1551, D1.13 p.p.m.). Low resolution LC-MS data were
measured using a Hewlett-Packard HP1100 integrated LC/MS system with
a reversed-phase C18 column (Phenomenex Luna, 4.6 mm  100 mm, 5 mm, Figure 1 Structure of marine-derived nosiheptide.8 A full color version of
Phenomenex, Torrance, CA, USA) at a flow rate of 0.7 ml min 1. this figure is available at The Journal of Antibiotics journal online.

Bacterial strains and susceptibility testing

Details on the strains used in this study are in Table 2. MRSA Sanger 252 USA
200 strain was obtained through the Network of Antimicrobial Resistance in
S. aureus (NARSA) program supported under NIAID/NIH contract no.
HHSN272200700055C. Susceptibility testing was performed in duplicate using
cation-adjusted Mueller–Hinton broth (CA-MHB) and Mueller–Hinton agar
according to Clinical and Laboratory Standards Institute methods.9 The MIC
for Clostridium difficile was determined by the agar dilution reference method
according to CLSI guidelines.10,11 Susceptibility of nosiheptide against MRSA
strain TCH1516 was also determined in the presence of 20% activated-pooled
human serum (freshly collected from consenting healthy donors) and 80% MHB

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 Nosiheptide activity against contemporary MRSA
NM Haste et al
595

Table 2 Nosiheptide MIC

Strain Details MIC (mg l 1)

TCH1516 USA 300 CA-MRSA (ATCC) 0.06 (0.06)a

Sanger 252 USA 200 HA-MRSA (NARSA) 0.03
ATCC 33591 HA-MRSA 0.06
A593720 Bloodstream MRSA, progenitor to A5940 0.125
A5940 VISA20 In vivo VISA 0.125
A630020 MRSA, progenitor to A6298 0.125
A6298 VISA20 VISA 0.125
SA85312 Progenitor to SI853b 0.125
SA853b12 In vitro VISA 0.125
HIP5836 NJ VISA12,20 VISA—NJ 0.125
VRSA PA12 vanA vancomycin-resistant Staphylococcus aureus 0.06
VRSA MI12 vanA vancomycin-resistant S. aureus 0.125
A781712 Progenitor to A7819, A7819erm 0.06
A7819LinR 12 LinezolidR isolate 0.06
A7819ermR 12 Erythromycin-resistant variant (plasmid) 0.06
SA35412 Progenitor to SA355 0.125
SA35512 LinezolidR isolate 0.125
061612 (DapS) Endocarditis MSSA, progenitor to 0701 0.125
070112 (DapR) Daptomycin-resistant MSSA 0.06
MSSA ATCC 29213 0.125
RN912020 MSSA, Agr knockout, progenitor to RN9120b 0.06
RN9120V20 VISA selected in vitro 0.25
Staphylococcus epidermidis ATCC 12228 0.5
VRE-CUS VRE bloodstream left-sided endocarditis 0.125
VRE-WMC VRE bloodstream infection BI/NAP1/027 Hypervirulent Contemporary 0.125
Clostridium difficile BI Clostridium difficile 0.008
Moraxella catarrhalis ATCC 25238 0.5
Eseudomonas coli 1035 21 Clinical respiratory tract isolate 44
Pseudomonas aeruginosa ATCC 27853 44
Acinetobacter baumannii ATCC 19606 44
Enterobacter cloacae ATCC 13047 44

Abbreviations: ATCC, American Type Culture Collection; VRE-CUS, vancomycin-resistant Enterococcus faecium.
aMIC in 20% human serum.

by adding resazurin and assessing the conversion to resorufin exactly as previ-
ously described.12 This method provides a visual readout of color change from
the blue indicator resazurin to the pink resorufin, a sign of bacterial growth.
Time-kill studies
Time-kill studies were done essentially as previously described in this
laboratory.13 Briefly, nosiheptide was added at multiples of the MIC in CA-
MHB. MRSA strains or vancomycin-resistant Enterococcus faecium were added
at a starting inoculum of 5  105 cfu ml 1, and the cultures were incubated in
a 37 1C shaking incubator (New Brunswick Scientific, Enfield, CT, USA).
Samples were taken at specified timepoints for enumeration of viable bacteria
by plating serial dilutions on Todd–Hewitt agar plates.
Mammalian cell cytotoxicity
Mammalian cell cytotoxicity was assessed as previously described.13 Briefly,
2  104 HeLa cells (American Type Culture Collection no. CCL-2, ATCC,
Manassas, VA, USA) were seeded per well of sterile 96-well tissue culture-
treated plates (Falcon; Becton Dickinson, Franklin Lakes, NJ, USA). After 24 h,
the medium (RPMI containing 10% heat-inactivated fetal bovine serum) was
replaced with fresh medium containing increasing concentrations of
nosiheptide up to 128 mg l 1, and the plates were incubated at 37 1C in 5%
CO2. Cell viability was assayed at 72 h by measuring the reduction of MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium) using the CellTiter 96 Aqueous nonradioactive cell
proliferation assay according to the manufacturer’s instructions (Promega,
Madison, WI, USA).

Post-antibiotic effect
PAE was assessed using the viable plate count method as previously
described.13 For these studies, either nosiheptide or vancomycin was added
at 10  their respective MICs to 14 ml Falcon tubes containing CA-MHB. CA-
MRSA strain TCH1516 or HA-MRSA strain Sanger 252 was added at 5  105
cfu ml, and the tubes were incubated under shaking conditions at 37 1C for 1 h.
The bacteria were then pelleted and washed in 4 ml of antibiotic-free CA-
MHB. The pellets were resuspended in 4 ml CA-MHB and incubated at 37 1C
in a shaking incubator. Surviving bacteria were assessed at specified timepoints
as for the time-kill assay. The PAE was determined as previously described.13,14
In vivo testing
A murine model of i.p. infection was used to test in vivo efficacy of
nosiheptide.15 Eight-week-old female CD1 mice (Charles River, Wilmington,
MA, USA) were injected i.p. with 1–2  109 cfu of the HA-MRSA strain Sanger
252 in 4% hog gastric mucin. The mice were treated with nosiheptide
(20 mg kg 1) i.p. at 1 and 8 h after bacterial inoculation and were monitored
for mortality and clinical status twice daily thereafter for 5 days. Moribund mice
were humanely killed as were mice at the end of the study. These experiments
were reviewed and approved by the Animal Subjects Committee of UCSD.

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 Nosiheptide activity against contemporary MRSA
NM Haste et al
596

Statistical analysis favorably with vancomycin kinetics at 10  and 20  their respective

The data from the survival studies were analyzed with GraphPad Prism
(GraphPad Software, Incorporated, La Jolla, CA, USA) using log-rank analysis
(Mantel–Cox test). Data were considered significant at Po0.05.
RESULTS
In vitro antimicrobial activity
Nosiheptide exhibited strong activity against all contemporary MRSA
and MSSA strains tested including multidrug-resistant clinical isolates
(Table 2), with MIC values p0.25 mg l 1. Notably, clinical MRSA
isolates previously demonstrated to have developed resistance to
front-line antibiotics including daptomycin, linezolid and vancomycin
remained highly sensitive to nosiheptide. This activity against MRSA
was also completely unaffected by the presence of 20% human serum
(Table 2). Nosiheptide was also highly active against Enterococcus spp.
and the Gram-negative pathogen Moraxella catarrhalis, but did not
possess activity at p4 mg l 1 against the other Gram-negative strains
tested including clinical isolates of Escherichia coli and Pseudomonas
aeruginosa. Addressing another critical antibiotic resistance challenge,
nosiheptide showed extremely potent activity against the contempor-
ary hypervirulent BI strain of C. difficile (also known as the NAP1 or
ribotype 027 strain).
Time-kill kinetics
In vitro time-kill analysis was used to assess nosiheptide killing
kinetics. Nosiheptide was rapidly bactericidal against MRSA in a
concentration- and time-dependent manner (Figure 2a), with a nearly
2-log kill noted within 6 h at both 10  and 20  MIC. A 2-log kill of
MRSA was also noted for nosiheptide against MRSA resistant to other
antibiotics, such as linezolid or erythromycin (Figure 2b). Although
nosiheptide demonstrated bactericidal activity against MRSA at 10 
and 20  the MIC, it was bacteriostatic against a clinical bloodstream
isolate of vancomycin-resistant Enterococcus faecium up to 64  MIC
(Figure 2c). The MRSA killing kinetics of nosiheptide also compared
MICs (Figure 2d).
Prolonged PAE and lack of cytotoxicity
Given its favorable MRSA killing kinetics, nosiheptide was assessed
for its PAE. Notably, nosiheptide exhibited prolonged PAEs against
both HA- and CA-MRSA compared with the most commonly used
systemic antibiotic, vancomycin, at 10  their respective MICs
(Figures 3a and b). The PAE for nosiheptide was calculated to exceed
9 h for both HA- and CA-MRSA. We also tested nosiheptide for
in vitro evidence of mammalian cell cytotoxicity as a prelude to our
in vivo experiments. No evidence for nosiheptide cytotoxicity was
observed when incubated for 72 h with the cervical carcinoma HeLa
cell line at up to 128 mg l 1, which is B1000-fold above the MIC
against MRSA. We also found that nosiheptide activity against
USA300 MRSA was not inhibited in 20% human serum (Table 2).
In vivo activity
Owing to its potent anti-MRSA activities in vitro, nosiheptide was
tested in a murine model of i.p. MRSA infection. Mice were infected
with HA-MRSA strain Sanger 252, followed by nosiheptide treatment
(20 mg kg 1, i.p.) at 1 and 8 h post infection. Although all mice
became lethargic and exhibited piloerection within B4 h of infection,
nosiheptide provided significant (Po0.03) protection against mor-
tality (Figure 4). Ten out of 10 of the nosiheptide-treated mice
remained alive on day 3, whereas 6/10 of the controls died on day 1.
By the end of the study, only one mouse in the nosiheptide group had
died. These results provide evidence of significant in vivo activity for
nosiheptide.
DISCUSSION
In our search for new anti-MRSA antibiotics from marine-derived
microorganisms, we identified an actinomycete strain that produced a
metabolite shown to be nosiheptide. Although the structure of this

10 10
None A7817
8 Nosiheptide 20X MIC 8 A7819
log10 cfu/ml

log10 cfu/ml

Nosiheptide 10X MIC A7819em

6 Nosiheptide 1X MIC 6 SI354
SI354-LinR
4 4 ATCC33591
2 2

0 0
0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24
Time (hr) Time (hr)

10 10
None None
8 Nosiheptide 2X MIC 8 Nosiheptide 20X MIC
log10 cfu/ml

log10 cfu/ml

Nosiheptide 4X MIC Vancomycin 20X MIC

6 6 Nosiheptide 10X MIC
Nosiheptide 8X MIC
Vancomycin 10X MIC
4 Nosiheptide 32X MIC 4
Nosiheptide 64X MIC
2 2

0 0
0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24
Time (hr) Time (hr)

Figure 2 Time-kill kinetics of nosiheptide against MRSA and VRE. Nosiheptide killing kinetics was studied against MRSA and VRE at multiples of their
MICs (MIC ¼ 0.06 mg l 1 for nosiheptide). (a) Killing kinetics of nosiheptide at 1  , 10  and 20X MIC against MRSA USA300 strain TCH1516
(MIC ¼ 0.06 mg l 1). (b) Nosiheptide killing kinetics against various multi-drug resistant MRSA at 10  their respective MICs. (c) Nosiheptide activity over
time against vancomycin-resistant Enterococcus faecium at multiples of the MIC (MIC ¼ 0.125 mg l 1). (d) Nosiheptide time-kill kinetics against MRSA
USA300 strain TCH1516 compared with vancomycin at 10  and 20  their respective MICs (MIC ¼ 0.06 mg l 1 for nosiheptide; MIC ¼ 1.56 mg l 1 for
vancomycin). Each assay was repeated in duplicate (for VRE) or triplicate (for MRSA), and the data shown are the mean±s.d. from one representative
experiment.

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 Nosiheptide activity against contemporary MRSA
NM Haste et al
597

CA-MRSA contemporary MRSA strains tested regardless of their resistance

to other front-line therapeutics including vancomycin, linezolid and
10 Nosiheptide 10X MIC daptomycin. Importantly, high-level activity was also noted against
8 Vancomycin 10X MIC the increasingly problematic pathogen, C. difficile, especially signifi-
log10 cfu/ml

None
cant as the FDA has approved only one new drug (Fidaxomicin) to
6
treat C. difficile in the past two decades. Although the activity of
4 some thiazole antibiotics may be reduced in the presence of serum,
2
we found that nosiheptide activity against USA300 MRSA was
unaffected in the presence of 20% human serum, suggesting that
0 serum inhibition may not necessarily be a characteristic of all
0 3 6 9 12 15 18 21 24 thiazole-containing antibiotics.
Time (hr) Nosiheptide exhibited favorable MRSA killing kinetics, a very
prolonged PAE, and was active in a murine model of i.p. infection.
HA-MRSA
Although in vivo evidence of nosiheptide activity has not been
10 published, Benazet et al.16 report that nosiheptide protected mice
Nosiheptide 10X MIC
8 Vancomycin 10X MIC from S. aureus mortality only when the compound was administered
at the site of infection. However, no data were presented to support
log10 cfu/ml

None
6 this claim. We report in the current paper nosiheptide activity when
4 the compound is administered 1 and 8 h after MRSA injection at the
site of infection as well (Figure 4). Our data are consistent with the
2 claims of Benazet et al.16 and point to the fact that nosiheptide is
0 efficacious in vivo but may either be metabolized or not distributed
0 3 6 9 12 15 18 21 24 (due to localized precipitation or lack of absorption) when injected. A
Time (hr) closely related compound, glycothiohexide a, was once shown to be
quite active against MRSA and vancomycin-resistant E. faecium.17–19
Figure 3 PAE of nosiheptide on contemporary MRSA strains. CA-MRSA
strain TCH-1516 (a) or HA-MRSA strain Sanger 252 (b) were incubated for
However, despite its in vitro potency, this compound had minimal
1 h with nosiheptide or vancomycin at 10  MIC. The antibiotics were activity in a S. aureus model of i.p. infection when administered s.c.
removed, and the bacteria were washed and allowed to recover in antibiotic- It is unknown whether in the case of glycothiohexide a, pharmaco-
free media. The curves show the rates of growth following antibiotic kinetic and/or possible metabolic factors abrogated potential efficacy.
treatment. The data represent the mean±s.d. of one representative assay In pilot studies, we also found that nosiheptide exhibited significantly
repeated in duplicate. reduced activity when injected s.c. (data not shown) following i.p.
MRSA inoculation and are currently embarking upon collaborative
studies of liposomal and other lipophilic drug formulations to
100 Nosiheptide optimize nosiheptide delivery in vivo.
Control In sum, nosiheptide represents a promising antibiotic scaffold for
80 further development owing to its potent anti-MRSA and other Gram-
% Survival

60
40
20
0 properties provide nosiheptide an advantage over other early-stage
0 1 2 3 4 5
Day
Figure 4 Nosiheptide protection in a murine model of i.p. MRSA infection.
Plot showing survival of mice (n ¼ 10 per group) infected i.p. with HA-
MRSA strain Sanger 252, followed by i.p. treatment with either nosiheptide
(20 mg kg 1, filled circles) or vehicle control (filled squares) at 1 h or 8 h
after infection (represented by upward arrows on the x axis). N ¼ 10 mice
per treatment group (Po0.03 by log-rank analysis).
compound was originally elucidated several decades ago and activity
against Gram-positive activity was noted, a paucity of data exist on
the therapeutic potential for this compound given contemporary
concerns of multi-drug resistant bacterial pathogens. With growing
antibiotic resistance exhibited by MRSA and other Gram-positive
pathogens driving the need for new antimicrobials, we sought
to further characterize this compound for its activity against
HA- and CA-MRSA. Nosiheptide was exquisitely active against all
positive activity, lack of inhibition by human serum, lack of
mammalian cell cytotoxicity and demonstrated tolerability when
delivered orally to animals.7 Many of the synthesis/manufacturing
issues have already been addressed, as nosiheptide is produced in
mass quantities by manufacturers worldwide. Cumulatively, these
antibacterials, many of which are later found to be cytotoxic or
serum-inhibited, or are fraught with difficulties in scale-up of
production.
ACKNOWLEDGEMENTS
NMH was supported by the National Institutes of Health (NIH) Training
Program in Marine Biotechnology (T32 GM067550) and a Ruth L Kirschstein
National Research Service Award (NRSA) from National Institutes of Health
grants (5 F31 GM090658-02). SL was supported by the German Research
Foundation (DFG). WF, VN, PRJ and MEH were supported by National
Institutes of Health grant GM084350. PRJ and WF acknowledge financial
support from the NIH Fogerty Center International Cooperative Biodiversity
Groups program (grant U01-TW00007-401). We thank K Freel and C
Kauffman for assistance with fieldwork and W Aalbersberg (University of the
South Pacific) for providing laboratory space and facilitating the field
collections. We gratefully acknowledge the people of Fiji for their hospitality
and permission to collect samples from their local waters. We are also grateful
to Diane M Citron and Ellie Goldstein MD of RM Alden Research
Laboratories (Culver City, CA, USA) for performing susceptibility testing
against Clostridium difficile.

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NM Haste et al
598
1 Dhand, A. et al. Use of antistaphylococcal beta-lactams to increase daptomycin activity
in eradicating persistent bacteremia due to methicillin-resistant Staphylococcus
aureus: role of enhanced daptomycin binding. Clin. Infect. Dis. 53, 158–163
(2011).
2 Bagley, M. C., Dale, J. W., Merritt, E. A. & Xiong, X. Thiopeptide antibiotics. Chem. Rev.
105, 685–714 (2005).
3 Nicolaou, K. C., Chen, J. S., Edmonds, D. J. & Estrada, A. A. Recent advances in the
chemistry and biology of naturally occurring antibiotics. Angew. Chem. Int. Ed. Engl.
48, 660–719 (2009).
4 Tanaka, T., Endo, T., Shimazu, A., Yoshida, R. & Suzuki, Y. A new antibiotic,
multhiomycin. J. Antibiot. 23, 231–237 (1970).
5 Tanaka, T., Sakaguchi, K. & Yonehara, H. On the mode of action of multhiomycin, I.
Effects of multhiomycin on macromolecular syntheses. J. Antibiot. 23, 401–407
(1970).
6 Cundliffe, E. & Thompson, J. The mode of action of nosiheptide (multhiomycin) and
the mechanism of resistance in the producing organism. J. Gen. Microbiol. 126,
185–192 (1981).
7 Benazet, F. & Cartier, J. R. Effect of nosiheptide as a feed additive in chicks on the
quantity, duration, prevalence of excretion, and resistance to antibacterial agents of
Salmonella typhimurium; on the proportion of Escherichia coli and other coliforms
resistant to antibacterial agents; and on their degree of resistance. Poult. Sci. 59,
1405–1415 (1980).
8 Mocek, U. et al. 1H and 13C NMR Assignments of the thiopeptide antibiotic
nosiheptide. J. Antibiot. 42, 1643–1648 (1989).
9 Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria that Grow Aerobically. Document M7–A7 (Clinical
and Laboratory Standards Institute, Wayne, PA, 2006).
10 Clinical and Laboratory Standards Institute. Methods for Antimicrobial Susceptibility
Testing of Anaerobic Bacteria. Approved Standard M11-A7 (Clinical and Laboratory
Standards Institute, Wayne, PA, 2007).
The Journal of Antibiotics
11 Goldstein, E. J. C. et al. Comparative susceptibilities to fidaxomicin (OPT-80) of
isolates collected at baseline, recurrence, and failure from patients in two Phase III
trials of fidaxomicin against Clostridium difficile infection. Antimicrob. Agents Che-
mother. 55, 5194–5199 (2011).
12 Sakoulas, G. et al. Novel bacterial metabolite merochlorin A demonstrates in vitro
activity against multi-drug resistant methicillin resistant Staphylococcus aureus. PloS
One 7, e29439 (2012).
13 Haste, N. M. et al. Pharmacological properties of the marine natural product
marinopyrrole A against methicillin resistant Staphylococcus aureus. Antimicrob.
Agents Chemother. 55, 3305–3312 (2011).
14 Craig, W. A. & Gundmundsson, S. in Antibiotics in Laboratory Medicine (ed. Lorian, V.)
4th edn. 296–329 (Williams and Wilkins, Baltimore, MD, 1996).
15 Haste, N. M. et al. Activity of the streptogramin antibiotic etamycin against methicillin-
resistant Staphylococcus aureus. J. Antibiot. 63, 219–224 (2010).
16 Benazet, F. et al. Nosiheptide, a sulfur-containing peptide antibiotic isolated from
Streptomyces actuosus 40037. Experientia 36, 414–416 (1980).
17 Northcote, P. T. et al. Glycothiohexide alpha, a novel antibiotic produced by ‘Sebekia’
sp., LL-14E605. II. Isolation and physical-chemical characterization. J. Antibiot. 47,
894–900 (1994).
18 Northcote, P. T., Siegel, M., Borders, D.B. & Lee, M.D. Glycothiohexide alpha, a novel
antibiotic produced by ‘Sebekia’ sp., LL-14E605. III. Structural elucidation.
J. Antibiot. 47, 901–908 (1994).
19 Steinberg, D. A. et al. Glycothiohexides, novel antibiotics produced by ‘Sebekia’ sp. LL-
14E605. I. Taxonomy, fermentation and biological evaluation. J. Antibiot. 47,
887–893 (1994).
20 Sakoulas, G. et al. Accessory gene regulator (agr) locus in geographically diverse
Staphylococcus aureus with reduced susceptibility in vancomycin. Antimicrob. Agents
Chemother. 46, 1492–1502 (2002).
21 Laplante, K. L. & Sakoulas, G. Evaluating aztreonam and ceftazidime pharmacodynamics
with Escherichia coli in combination with daptomycin, linezolid, or vancomycin in an
in vitro pharmacodynamic model. Antimicrob. Agents Chemother. 53, 4549–4555 (2009).